Background[unreadable] [unreadable] Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by self-limited attacks of fever with serosal, synovial, or cutaneous inflammation, sometimes complicated by systemic amyloidosis. In 1992 our laboratory mapped the FMF locus to chromosome 16p13.3, and in 1997 we isolated the underlying gene, MEFV, and demonstrated that it is highly expressed in granulocytes. We have subsequently studied FMF population genetics, the regulation of FMF gene expression in leukocyte subpopulations, the biochemistry and cell biology of pyrin (the FMF protein), and the development of animal models of FMF. The N-terminal 90 amino acids of pyrin comprise a motif, commonly called the PYRIN domain, found in approximately 20 human proteins. The cognate interaction of the PYRIN domain of pyrin with the homologous domain of the apoptosis-associated specklike protein with a CARD (ASC) places pyrin upstream in the regulation caspase-1-mediated interleukin-1 (IL-1) beta activation. In studies of peritoneal macrophages from a mouse strain expressing a truncated form of pyrin, we found increased caspase-1 activation and IL-1 beta processing, and impaired apoptosis through a caspase-8-dependent, IL-1 beta-independent pathway. By yeast two-hybrid studies, we demonstrated that pyrin interacts with the cytoskeletal protein PSTPIP1, and that PSTPIP1 mutations associated with the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA) lead to markedly increased pyrin-binding and IL-1 beta activation. [unreadable] [unreadable] Results of the Last Year[unreadable] [unreadable] Cleavage of pyrin by caspase-1: During the current reporting period we completed and published a study that follows up on our previous observations that the C-terminal B30.2 domain of pyrin interacts directly with the catalytic p20 and p10 domains of caspase-1. We found that pyrin is cleaved by caspase-1 at Asp330, a site remote from the B30.2 domain. Pyrin variants harboring the FMF-associated M694V, M680I, and V726A mutations were cleaved more efficiently than wild-type pyrin. Further studies, summarized in last years report, implicate the N-terminal cleaved fragment of pyrin in NF-kappa B activation. [unreadable] [unreadable] Studies of murine models of FMF: During the last year we have continued our studies of pyrin full knockout mice, which follow up on the aforementioned characterization of pyrin truncation mice, and have begun full-scale characterization of knockin (KI) mice for the V726A, M680I, and M694V mutations. The homozygous KI mice with FMF mutations showed severe inflammatory phenotypes, such as eye inflammation, skin rash, hair loss, arthritis, and reduced body size. Mutant B30.2 domain KI mice also manifested splenomegaly and lymphomegaly, largely due to large numbers of CD11b macrophages and granulocytes. KI mice also exhibited significantly increased serum cytokines, including IL-1beta, IL-6, TNF-alpha, GM-CSF, and KC. The V726A KI mice showed the most severe phenotype and the M694V showed a mild and delayed phenotype, while the M680I showed an intermediatry phenotype. This contrasts with the mutations in humans, where the M694V is generally the most severe and V726A the mildest. Homozygotes for the wild type human B30.2 domain were embryonic lethal. All of the heterozygous KIs are healthy, even the V726A/null hemizygotes. These results suggest a gain-of-function model with a dosage effect. Additional experiments crossing the KIs with IL-1 receptor knockout mouse indicate a marked attenuation of the phenotype, thus demonstrating a major role for IL-1 in disease pathogenesis. Bone marrow transplantation suggests a predominant role for hematopoietic lineages in disease pathogenesis in the KI mice.[unreadable] [unreadable] RNA interference of MEFV in human monocytic cells: We continued studies of siRNA-mediated inhibition of MEFV in human THP.1 monocytic cell lines, described in the previous years report. New studies included functional assays, demonstrating decreased internalization of E. coli bioparticles by phagocytosis in siMEFV treated cells. Because previous studies of cell lines implicated IRF2 as an intermediary in the pyrin pathway in THP.1 cells, we studied IRF2 message expression in leukocytes from FMF patients on and off colchicine relative to controls, and found evidence that mutations in MEFV may in some cases behave like the knockdown in THP.1 cells. We are currently preparing a manuscript summarizing these findings.[unreadable] [unreadable] Mutational studies in patients: We continued studies begun last year in patients with clinical FMF but only one demonstrable MEFV mutation. We substantially expanded the number of patients studied during the past year to 44. Using conventional capillary electrophoresis, we screened all of these patients for a second mutation in the coding region, but failed to find any. The entire 15 kb MEFV genomic region was resequenced in 10 patients using a hybridization-based chip technology, again without finding a likely disease-asociated mutation. Western blots did not demonstrate a significant difference in pyrin levels between single and double variant patients; however, FMF patients of both types showed higher protein expression compared to controls. Mutational screening of genes encoding pyrin-interacting proteins identified rare variants in a number of patients, such as the V120M variant of SIVA and the W171X variant of ASC. Although the functional consequences of these variants are still unknown, these data suggest the possibility of digenic inheritance. [unreadable] [unreadable] Development of animal models for PAPA syndrome: During the previous reporting period we have developed PSTPIP1 knockout mice, and knockins for the PAPA-associated E250Q mutation. Phenotypic analyses are currently under way.[unreadable] [unreadable] Discovery of a novel recessively-inherited autoinflammatory syndrome: In collaboration with Dr. Goldbach-Mansky our laboratory discovered a new illness characterized by the total absence of the interleukin-1 receptor antagonist. To date we have observed this recessively inherited condition, which we term DIRA (deficiency of the IL-1 receptor antagonist), in three unrelated families from Newfoundland, the Netherlands, and Puerto Rico. The Canadian family harbors a two base-pair deletion (N52Kfs25), and the Dutch family a nonsense mutation (E77X), both leading to premature truncation of the protein. Neither mutation was observed in large panels of control chromosomes. In contrast, the Puerto Rican family manifests an approximately 170 kb genomic deletion of the region on chromosome 2 that encodes not only the IL-1 receptor antagonist gene (IL1RN), but five other molecules in the IL-1 family. Molecular analyses demonstrate the total absence of IL1RN message and protein in affected individuals from all three families, with intermediate levels in heterozygotes. When blood from affected individuals was cultured with bacterial LPS, there were significant increases in the production of a number of cytokines, including IL-1&#945;, MIP-1alpha, TNF-alpha, IL-6, and IL-8, thus indicating that the absence of a key regulator of IL-1beta leads to far-reaching activation of innate immunity. The clinical implications of unchecked IL-1 signaling are similarly impressive, with patients manifesting, among other things, diffuse pustulosis, cystic bone lesions, and vasculitis. These data suggest previously unrecognized ramifications of IL-1 activation in other human disorders. Supporting this view, DIRA patients manifest a dramatic clinical response to anakinra, the recombinant IL-1 receptor antagonist. [unreadable] [unreadable] Conclusions and Significance: Data obtained during the current reporting period extend our understanding of the molecular basis of human autoinflammatory/ autoimmune disease. During the next year we intend to extend mechanistic studies of known diseases, as well as continuing our search for novel genes.